Many electrochemical biosensors for point of use applications have been developed over the last 30 years, and a common goal for such sensors is to achieve a fast response, preferably with single use device that makes possible the selective quantification of a target analyte in a complicated matrix without sample pre-treatment.
For example, amperommetric glucose biosensors are the most commercially successful products to result from this research and development effort. Besides such glucose biosensors, several other oxidase-based biosensors have been developed for medical and environmental applications. However, field application of biosensors based on reductases is often limited by the need for anaerobic conditions. For example, the reduction of nitrate catalyzed by nitrate reductase occurs at potentials more negative than the oxygen reduction reaction, and oxygen must therefore be removed from the sample before analysis. While nitrate biosensors for on-site determination of nitrate in water, soil or plant samples is potentially very valuable with respect to economic, environmental, and health issues, standard oxygen removal methods based on argon purging or vacuum degassing are not compatible with on-site monitoring. Such need for oxygen removal is not limited to nitrate reductases only, but is also generally required for numerous other reductase-based biosensors.
Recently the use of sodium sulfite as an oxygen scavenger for nitrate amperommetric biosensor based on recombinant eukaryotic nitrate reductase was described. However, the maximum sulfite concentration which does not affect the nitrate analysis is about 1 mM, which is often sufficient for large sample volumes or closed systems. However, in open systems (e.g., systems where the sample is exposed to atmospheric oxygen) or systems with sample volumes of 200 μL or less, it is typically not possible to maintain anaerobic conditions with sulfite for the time required for electrochemical measurements. Similar difficulties may be encountered in the system and methods described in U.S. Pat. No. 2,482,724 where an enzymatic system is used to reduce oxygen from food stuff in a closed container. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
The most recent advance in biosensor technology is the “electron-carrier free” system, where an enzyme is directly immobilized on the working electrode of the electrochemical cell (see e.g., U.S. Pat. No. 4,970,145). This may result in direct electron transfer from the electrode to the electron-carrying group of the enzyme, such that the enzyme is “wired” to the electrode. In this system, interference by oxygen is minimized unless the immobilized, reduced reductase reacts with oxygen. However, if the potential required to drive the detection reaction is more negative than the potential required for reducing oxygen (which may depend on the electrode material), then oxygen interference may occur. In these cases, an oxygen removal system will still be required to facilitate operation of the wired reductase biosensor.
Remarkably, the need for oxygen removal in solution extends well beyond the enzyme biosensors, and in fact, may be present in numerous alternative electrochemical systems. For example, various biofuel cells are operated under anaerobic conditions in some formulations (see e.g., US2010/0297737). In such case, an oxygen removal system will typically be required to facilitate operation of the biofuel cell.
Thus, even though various systems and methods are known to reduce oxygen in a liquid reaction system, all or almost all of them suffer from one or more disadvantage. Therefore, there is still a need to provide improved oxygen removal systems suitable for use with biosensors and other electrochemical reaction systems.